VIRAL VECTOR AND siRNA CORE FACILITY 1. MAIN OBJECTIVES AND NEW DIRECTIONS The Viral Vector/siRNA Core will be a new facility. Currently, there is no unified Core facility to link neuroscientists on the La Jolla Torrey Pines Mesa for these functions. Therefore, this facility will constitute a new Core to foster the use of viral vectors for payload delivery into neural cells, including RNAi, among neuroscientists and will produce multiple collaborations in an interdisciplinary manner in the San Diego area on the La Jolla Torrey Pines Mesa. The primary core will be located at The Salk Institute because of the presence of a prominent virologist/geneticist there, Dr. Inder Verma, who will Direct the Core with the assistance of Edward Callaway, who will act as Co-Director. A satellite facility will be located at the Burnham Institute for Medical Research (BIMR) headed by Mark Mercola, who will serve as a Co-Director for the core. This satellite is particularly important for seamless interface with the other cores located there that will use the viral vectors, e.g., the Chemical Library Screening/High Throughput Cell Analysis Cores and the Imaging Core. The mission of the Viral Vector and siRNA Core Facility (WS) is to provide a robust means of gene manipulation to Neuroscience investigators. Over the last decade, methods for the production of a variety of viral vectors for stable and non-toxic delivery of genetic material to postmitotic neurons have become relatively routine. However, the laboratories that would most benefit from the use of these vectors often have expertise in neuroanatomy, neurophysiology, or behavior, but not virology. This disparity between expertise and need greatly limits the use of these valuable vectors within the San Diego Neuroscience community. Furthermore, even those laboratories that have the technical capability to produce one or another type of vector would benefit from a core facility which maintains proven protocols, technical expertise and experience, and all the necessary plasmids required to implement the full range of possibilities afforded by modern virology. The San Diego neuroscience community presently has the expertise to establish and maintain a state-of-the art facility, but piecemeal collaboration amongst a limited set of laboratories does not fully realize the potential of this community. Establishing a facility to make a broad range of vectors including adeno-associated virus (AAV), lentivirus, and herpes simplex virus (HSV) amplicon vectors will have fundamental impact on the culture of Neuroscience in San Diego. This core will make possible experiments that would be otherwise impossible and will foster collaborations amongst molecular neuroscientists who routinely use genetic methods, and other neuroscientists who focus on behavior, anatomy or physiology. Viral vectors are now fundamental tools at the cutting edge of experimental neuroscience. This is because they harness the proven power of molecular and genetic techniques by complementing and extending the capabilities afforded by the production of transgenic mice. Amongst the most prominent advantages of viral vectors are: 1) the short time required to test a genetic manipulation (e.g. gene knockdown with siRNA) compared to the generation of a transgenic mouse line;2) the ability to target gene expression to particular brain regions where specific promoters are not available for targeting;3) the ability to initiate manipulation in adult animals;4) the ability to use genetic technologies in species where transgenic methods are impractical (e.g., monkeys). Nevertheless, each of the available viral vector technologies have limitations, such that no single vector will meet the needs of every possible experimental goal. We have selected three types of viral vectors, AAV, lentivirus, and HSV amplicons, because these are all proven to be highly effective and they are a complementary set. Limitations of one vector can often be obviated by the use of another. These vectors all have in common, however, that they can efficiently transduce nearly every neuron in the vicinity of a brain injection and they can yield stable gene expression for months or years without toxicity (Naldini et al., 1996;Blomer et al., 1997;Xiao et al., 1997;Rabinowitz and Samulski, 1998;Sandier et al., 2002;Davidson and Breakefield, 2003; Kootstra and Verma, 2003). In addition to supporting the construction and use of viral vectors, the core will also provide reagents and expertise in gene knockdown by si/shRNA. Gene attenuation has been central to experimental biology for the past century and has increased exponentially with the advent of reverse genetics or the use of targeted attenuation of gene activity to reveal function. It is anticipated that gene attenuation studies performed at a large, genome or proteome-wide scale, will be increasingly important to assign gene function now that the sequences of many experimental organisms are complete. Moreover, gene function will point to targets that will be interrogated further using other technologies embodied in the other Neuroscience Cores, including Chemical Library Screening and Structural Biology;thus, this core is an important component of an integrated approach to Neurosciences. Currently, there is no si/shRNA and viral vector facility or core service available to Neuroscientists. Individual laboratories contract with commercial entities to produce siRNA oligos or construct si or shRNA vectors internally and viral vector technology is represented at a very sophisticated level in a few laboratories. The Cancer Center at BIMR is establishing a facility that will distribute a commercial library of siRNA oligonucleotides against human genome targets, but this will be available only to Cancer Center members, and efficacy of transfection of siRNA oligonucleotides into many cell lines, especially primary cell lines, is limited. The core facility will offer 1) AAV, lenti and HSV vector support, 2) access to a commercial siRNA oligonucleotide library for screening, 3) access to a shRNA lentiviral library to the human kinome, 4) . shRNA lentiviral libraries to other targets will be offered as libraries become available;in addition, the core will offer: 5) tagged human cDNA clones representing the entire kinome in a 3rd generation lentiviral vector and 6) a negotiated preferred price structure for purchasing siRNA oligonucleotides from a commercial vendor. Kinases have been chosen for the first shRNA and expression lentiviral libraries to be offered by the facility because they constitute one of the largest and most important of protein families, accounting for ~2% of genes in human and other eukaryotic genomes (Manning et al., 2002a;Manning et al., 2002b). By phosphorylating substrate proteins, kinases modify the activity, location, and affinities of up to 30% of all cellular proteins, and direct most cellular processes, particularly in signal transduction and coordination of complex pathways. In fact, it is difficult to think of any pathway that is not modulated by kinases, making them, as a group, a lynchpin of cell biology, and promising that a set of high quality knockdown reagents would provide insight into the control of almost any cellular process. The human genome contains 518 kinase genes, of which over 150 have already been implicated in human disease (Manning, 2005). The WS facility will be housed at the Salk Institute with a satellite facility at the BIMR. The purpose of the BIMR satellite is to have access to the robotic liquid handling capacity of the Chemical Library Screening (CLS) to amplify and array the libraries. The Salk facility will be under the direction of Inder Verma, PhD and Ed Callaway, PhD as co-director. Dr. Verma's laboratory has dedicated the last 30 years to understanding the molecular underpinnings that convert a normal cell to a cancer cell. He has developed expertise in large-scale manipulation of lentiviral vectors for the expression of cDNAs and si/shRNA. Dr. Callaway is an expert in cortical neural function and organization and his lab has expertise in the production and use of AAV, lentiviral and HSV amplicon vectors for gene delivery. The lab also has extensive experience in using these vectors in vivo in monkeys, ferrets, rats and mice.